This project addresses research questions pertinent to the very initial stages of tooth enamel formation and the mechanisms of protein-mediated control of spatial organization and phase of forming calcium phosphate, the mineral in our bones and teeth. We propose a biomimetic approach to test the working hypothesis that phosphorylated full-length amelogenin specifically regulates the initial formation of parallel arrays of apatitic crystals in vivo. Furthermore, this project aims to develop the use of Helium Ion Microscopy (HIM) for the high-resolution visualization and analysis of both mineral and organic phase in samples from: (i) in vitro mineralization studies and (ii) developing teeth from mice. Due to the novelty of the analytical approach of HIM for the study of biomineralization, we will work in close collaboration with the microscope manufacturer Carl Zeiss, NTS LLC., to fully exploit the outstanding and unique features of HIM for biomedical research. The Specific Aims are 1) To test the hypothesis that the full-length amelogenin forms unique protein-mineral assemblies that regulate the structural organization and result in parallel arrays of apatitic crystals in vitro similar to those seen in developing enamel. More specifically, we will apply HIM analyses to samples from in vitro mineralization experiments using mixtures of full-length and cleaved forms of pig amelogenin to critically test this hypothesis using various substrate surfaces which may influence protein assembly. Recombinant (rP172 and rP147) amelogenins will first be used to optimize HIM protocols for simultaneous characterization of both protein and mineral phases at sub-nanometer resolution to then also study the analogous native proteins (P173 and P148). 2) To optimize the use of fluorescent markers with HIM analyses to test the hypothesis that full-length and cleaved amelogenins do not co-localize on mineral phases formed in the presence of their mixture, but rather each protein exhibits a distinctive self-assembly pattern and distribution relative to each other and the mineral phase. Specifically, we will apply fluorescent markers with HIM analyses a) first for in vitro samples from mineralization experiments comprised of different labeled enamel matrix proteins/peptides (rP172, rP147, LRAP) and mineral phases, and then b) for developing mouse enamel incisors examined during the secretory stage of amelogenesis. In particular, we will use primary and secondary antibodies for the specific labeling of enamel matrix proteins and/or their degradation products to allow for the identification and location of full-length and cleaved amelogenins and/or other enamel matrix proteins (LRAP, enamelin) in vitro and in situ. These studies will again be carried out in collaboration with Carl Zeiss, SMT. Achieving these goals will elucidate fundamental mechanisms of protein-guided mineralization and the role of specific enamel matrix proteins in regulating crystal shape and alignment during amelogenesis. The innovative use of HIM for fluorescence imaging introduces a new imaging technique into biomineralization studies for direct visualization of protein-mineral relationships in both wild type and knock out animals.